Potent inositol 1,4,5-trisphosphate (IP3) receptor agonists, adenophostin A and B, were found from fungal products. In spite of the striking structural difference from IP3, adenophostins were found to inhibit [3H]IP3 binding more potently than IP3: the Ki values for adenophostin A and B were both calculated to be 0.18 nM, while that of IP3 was 15 nM. Adenophostins induced Ca2+ release both from cerebellar microsomes and from intracellular Ca2+ stores in permeabilized NG108-15 cells. Adenophostins at concentration as low as 1 nM produced a significant Ca2+ release from cerebellar microsomes, and their activities were 100-fold more potent than IP3. In addition, heparin, an IP3 receptor antagonist, completely blocked the Ca2+ releasing activity of adenophostins. Adenophostins were resistant to phosphorylation and dephosphorylation by IP3-metabolizing enzymes, thereby providing a possible explanation for their prolonged activities. Adenophostin also bound to plasma membrane IP3 receptor with a high affinity and inhibited [3H]IP3 binding to jurkat human T-cell plasma membranes: the IC50 value for adenophostin A was 0.95 nM. Adenophostin may prove to be a powerful tool for investigating the physiological properties of IP3 and its receptor.